|
| Status |
Public on Aug 08, 2024 |
| Title |
dog_2_b_ucc_replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
SEC fraction b
|
| Organism |
Canis lupus familiaris |
| Characteristics |
tissue: SEC fraction b
genotype: urothelial carcinoma
|
| Treatment protocol |
Protease inhibitor was added in to melted urine, urine was centrifuged at 4000 x g, for 20 minutes at 4 °C, and the supernatant was filtered using 0.22µm syringe PES filters (Millex GP, #SLGP033RS) and concentrated to <130µL using Amicon Ultra centrifugal ultrafiltration columns (100 kDa cut off, #UFC810024, Millipore). The concentrated urine was fractioned using qEV single 70nm SEC columns (Izon Science) and fractions b (EVs) and d (proteins) were used in RNA extraction.
|
| Growth protocol |
The urine was collected, centrifuged at 2000 x g for 10 minutes (to pellet cellular components) and the cell-free supernatant was frozed at -80C until batch analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using miRNeasy Mini Kit (#217004 Qiagen) from 500ul of EV and protein fractions.
Libraries were prepared using QiaSeq miRNA library kit (#331505, Qiagen) combined with QIAseq miRNA 48 Index IL kit (#331595, Qiagen). "miRNA" library (SLX-19754) was prepared according to the suggested manufacturer protocol. "Long RNA" library (SLX-19755) was acquired by performing the wash steps to the beads that were meant to be discarded in the first bead purufication step of the official protocol.
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
MIHT29_002_b_ucc
|
| Data processing |
In command line, saving lines from fastq files including the middle "AACTGTAGGCACCATCAAT" adapter using "zgrep" into a new text file
Using a self-written R-script, generating a table of individual sequences and their count number in each sample using the Unique Molecular Identifiers.
Aligning the sequences to canine mature miRNAs using blast from DEUS pipeline 1.0 with one mismatch and combining the count numbers for sequences that aling to same miRNA.
Assembly: miRbase release 22.1
Supplementary files format and content: csv file including the count numbers for mature miRNAs for each sample
|
| |
|
| Submission date |
Feb 23, 2024 |
| Last update date |
Aug 08, 2024 |
| Contact name |
Lajos Kalmar |
| E-mail(s) |
lk397@cam.ac.uk
|
| Phone |
+441223335457
|
| Organization name |
MRC Toxicology Unit, University of cambridge
|
| Street address |
Tennis Court Road
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 1QR |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL25760 |
| Series (1) |
| GSE256461 |
miR-182, miR-221 and miR-222 are potential urinary extracellular vesicle biomarkers for canine urothelial carcinoma |
|
| Relations |
| BioSample |
SAMN40078848 |
| SRA |
SRX23718086 |
